Bacillus pumilus laccase: a heat stable enzyme with a wide substrate spectrum

Abstract

Background: Laccases are multi-copper oxidases that catalyze the one electron oxidation of a broad range of compounds. Laccase substrates include substituted phenols, arylamines and aromatic thiols. Such compounds are activated by the enzyme to the corresponding radicals. Owing to their broad substrate range laccases are considered to be versatile biocatalysts which are capable of oxidizing natural and non-natural industrial compounds, with water as sole by-product.

Results: A novel CotA-type laccase from Bacillus pumilus was cloned, expressed and purified and its biochemical characteristics are presented here. The molecular weight of the purified laccase was estimated to be 58 kDa and the enzyme was found to be associated with four copper atoms. Its catalytic activity towards 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), 2,6-dimethoxyphenol (2,6-DMP) and syringaldazine (SGZ) was investigated. The kinetic parameters KM and kcat for ABTS were 80 ± 4 μM and 291 ± 2.7 s(-1), for 2,6-DMP 680 ± 27 μM and 11 ± 0.1 s(-1) and for SGZ only kcat could be estimated to be 66 ± 1.5 s(-1). The pH optimum for ABTS was 4, for 2,6-DMP 7 and for SGZ 6.5 and temperature optima for ABTS and 2,6-DMP were found to be around 70°C. The screening of 37 natural and non-natural compounds as substrates for B. pumilus laccase revealed 18 suitable compounds. Three of them served as redox mediators in the laccase-catalyzed decolorization of the dye indigocarmine (IC), thus assessing the new enzyme's biotechnological potential.

Conclusions: The fully copper loaded, thermostable CotA laccase from Bacillus pumilus is a versatile laccase with potential applications as an industrial biocatalyst.

Figure 1

SDS-PAGE analysis of CotA purification. Samples from cell free extracts (CFE), heat incubation (HI), anion exchange chromatography (AEC) and size exclusion chromatography (SEC) were denatured for 20 min at 95°C in SDS loading buffer containing β-mercapthoethanol. Sample SEC* was incubated for 5 min at 95° in SDS loading buffer containing β-mercapthoethanol. M, marker. Specific activity was determined using the standard ABTS oxidation assay.

Figure 2

Effect of DMSO upon CotA activity from B. pumilus. Effect of DMSO present in the reaction mixture upon initial rate of SGZ (•), ABTS (▼) and 2,6-DMP (○) oxidation. Relative activity was normalized to the maximal activity achieved for each substrate. All data points represent mean values from triplicate determinations.

Figure 3

IC transformation. Time course of IC degradation by purified laccase from B. pumilus monitored spectroscopically at λ = 650 nm over a total of 48 h at 37°C. Reactions were conducted at pH 6.5 and 7.8, in the presence or absence of ACS. (A) 0.25 mM IC and ACS added at a ratio of 1:100. (B) 1 mM IC and ACS added at a ratio 1:100. (C) 0.25 mM IC and ACS added at a ratio of 1:1000. (D) 1 mM IC and ACS added at a ratio of 1:1000.